Novel genes and proteins encoded thereby

ABSTRACT

The present invention encompasses novel mammalian cell cycle checkpoint genes/DNA repair genes, cDNA or genomic DNA, isolated nucleic acids corresponding thereto, proteins encoded thereby, expression vectors comprising said nucleic acids, host cells transformed with said expression vectors, and methods for treating a cell using such nucleic acids or proteins.

REFERENCE TO RELATED APPLICATIONS

The present application is a division of U.S. patent application Ser.No. 10/229,355, filed on Aug. 27, 2002, now U.S. Pat. No. 7,214,790,which is a division of U.S. patent application Ser. No. 09/661,711,filed on Sep. 14, 2000, now U.S. Pat. No. 6,440,732, which claims thebenefit of U.S. Provisional Application for patent Ser. No. 60/153,836,filed on Sep. 14, 1999, each of which is incorporated herein byreference.

GOVERNMENTAL RIGHTS

This invention was made with government support under Grant Nos. CA77325and GM19234 from the National Institutes of Health. The United Statesgovernment has certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates generally to the field of medicine, andrelates specifically to methods and compositions for modulating cellgrowth and death, including cell formation of tissues, using novelproteins, variants of these proteins and nucleic acids encoding them.

BACKGROUND OF THE INVENTION

The integrity of the genome is of prime importance to a dividing cell.In response to DNA damage, eukaryotic cells rely upon a complex systemof controls to delay cell-cycle progression. The normal eukaryoticcell-cycle is divided into 4 phases (sequentially G1, S, G2, M) whichcorrelate with distinct cell morphology and biochemical activity. Cellswithdrawn from the cell-cycle are said to be in G0, or non-cyclingstate. When cells within the cell-cycle are actively replicating,duplication of DNA occurs in the S phase, and active division of thecell occurs in M phase. See generally Benjamin Lewin, GENES VI (OxfordUniversity Press, Oxford, GB, Chapter 36, 1997). DNA is organized in theeukaryotic cell into successively higher levels of order that result inthe formation of chromosomes. Non-sex chromosomes are normally presentin pairs, and during cell division, the DNA of each chromosomereplicates resulting in paired chromatids. (See generally BenjaminLewin, GENES VI (Oxford University Press, Oxford, GB, Chapter 5, 1997).

The eukaryotic cell cycle is tightly regulated by intrinsic mechanismsthat ensure ordered progression through its various phases andsurveillance mechanisms that prevent cycling in the presence of aberrantor incompletely assembled structures. These negative regulatorysurveillance mechanisms have been termed checkpoints (Hartwell andWeinert, 1989, “Checkpoints: controls that ensure the order of cellcycle events” Science, 246: 629-634). The mitotic checkpoint preventscells from undergoing mitosis until all chromosomes have been attachedto the mitotic spindle whereas the DNA structure checkpoint, which canbe subdivided into the replication and DNA damage checkpoint, result inarrests at various points in the cell cycle in the presence of DNAdamage or incompletely replicated DNA (Elledge, 1996, “Cell cyclecheckpoints: preventing an identity crisis.” Science 274: 1664-1672).These arrests are believed to allow time for replication to be completedor DNA repair to take place. Cell cycling in the presence of DNA damage,incompletely replicated DNA or improper mitotic spindle assembly canlead to genomic instability, an early step in tumorigenesis. Defectivecheckpoint mechanisms, resulting from inactivation of the p53, ATM, andBub1 checkpoint gene products have been implicated in several humancancers.

Checkpoint delays provide time for repair of damaged DNA prior to itsreplication in S-phase and prior to segregation of chromatids in M-phase(Hartwell and Weinert, 1989, supra.). In many cases the DNA-damageresponse pathways cause arrest by inhibiting the activity of thecyclin-dependent kinases (Elledge, 1997, supra.). In human cells theDNA-damage induced G2 delay is largely dependent on inhibitoryphosphorylation of Cdc2 (Blasina et al., 1997, “The role of inhibitoryphosphorylation of cdc2 following DNA replication block and radiationinduced damage in Human cells.” Mol. Biol. Cell 8: 1013-1023; Jin etal., 1997, “Role of inhibiting cdc2 phosphorylation in radiation-inducedG2 arrest in human cells.” J. Cell Biol., 134: 963-970), and istherefore likely to result from a change in the activity of the opposingkinases and phosphatases that act on Cdc2. However, evidence that theactivity of these enzymes is substantially altered in response to DNAdamage is lacking (Poon et al., 1997, “The role of cdc2 feedback loopcontrol in the DNA damage checkpoint in mammalian cells.” Cancer Res.,57: 5168-5178).

Three distinct Cdc25 proteins are expressed in human cells. Cdc25A isspecifically required for the G1-S transition (Hoffmann et al., 1994,“Activation of the phosphatase activity of human CDC25A by a cdk2-cyclinE dependent phosphorylation at the G-1/S transition.” EMBO J., 13:4302-4310; Jinno et al., 1994, “Cdc25A is a novel phosphatasefunctioning early in the cell cycle” EMBO J., 13: 1549-1556), whereasCdc25B and Cdc25C are required for the G2-M transition (Gabrielli etal., 1996, “Cytoplasmic accumulation of cdc25B phosphatase in mitosistriggers centrosomal microtubule mucleation in HeLa cells” J. Cell Sci.,109(5): 1081-1093; Galaktionov et al., 1991, “Specific activation ofcdc25 tyrosine phosphatases by B-type cyclins: evidence for multipleroles of mitotic cyclins” Cell, 67: 1181-1194; Millar et al., 1991,“p55CDC25 is a nuclear protein required for the initiation of mitosis inhuman cells” Proc. Natl. Acad. Sci. USA, 88: 10500-10504; Nishijima etal., 1997, J. Cell Biol., 138: 1105-1116). The exact contribution ofCdc25B and Cdc25C to M-phase progression is not known.

Much of our current knowledge about checkpoint control has been obtainedfrom studies using budding (Saccharomyces cerevisiae) and fission(Schizosaccharomyces pombe) yeast. A number of reviews of our currentunderstanding of cell cycle checkpoint in yeast and higher eukaryoteshave recently been published (Hartwell & Kastan, 1994, “Cell cyclecontrol and Cancer” Science 266: 1821-1828; Murray, 1994, “Cell cyclecheckpoints” Current Opinions in Cell Biology, 6: 872-876; Elledge,1996, supra; Kaufmann & Paules, 1996, “DNA damage and cell cyclecheckpoints” FASEB J., 10: 238-247). In the fission yeast six geneproducts, rad1⁺, rad3⁺, rad9⁺, rad17⁺, rad26⁺, and hus1⁺ have beenidentified as components of both the DNA-damage dependent andDNA-replication dependent checkpoint pathways. In addition cds1⁺ hasbeen identified as being required for the DNA-replication dependentcheckpoint and rad27+/chk1⁺ has been identified as required for theDNA-damage dependent checkpoint in yeast.

Several of these genes have structural homologues in the budding yeast.Further conservation across eukaryotes has recently been suggested withthe cloning of several human homologues of S. pombe checkpoint genes,including two related to S. pombe rad3⁺: ATM (ataxia telangiectasiamutated) (Savitsky et al., 1995, “A single ataxia telangiectasia genewith a product similar to PI-3 kinase” Science, 268: 1749-1753) and ATR(ataxia telangiectasia and rad3⁺ related)(Bentley et al., 1996, “TheSchizosaccharomyces pombe rad3 checkpoint genes” EMBO J., 15: 6641-6651;Cimprich et al., “cDNA cloning and gene mapping of a candidate humancell cycle checkpoint protein” 1996, Proc. Natl. Acad. Sci. USA 93:2850-2855); and human homologues of S. pombe rad9+, Hrad9 (Lieberman etal., 1996, “A human homolog of the Schizosaccharomyces pombe rad9+checkpoint control gene” Proc. Natl. Acad. Sci. USA 93: 13890-13895),Hrad1(Parker et al., 1998, “Identification of a human homologue of theSchizosaccharomyces pombe rad17+ checkpoint gene” J. Biol. Chem.273:18340-18346; Freire et al., 1998, “Human and mouse homologs ofSchizosaccharomyces pombe rad1(+) and Saccharomyces cerevisia RAD17:linkage to checkpoint control and mammalian meiosis” Genes Dev.12:2560-2573; Udell et al., 1998, “Hrad1 and Mrad1 encode mammalianhomologues of the fission yeast rad1(+) cell cycle checkpoint controlgene” Nucleic Acids Res. 26:2971-3976), Hrad17 (Parker et al., 1998,supra), Hhus1 (Kostrub et al., 1998, “Hus1p, a conserved fission yeastcheckpoint protein, interacts with Rad1p and is phosphorylated inresponse to DNA damage” EMBO J. 17:2055-2066), Hchk1 (Sanchez et al.,1997, “Conservation of the Chk1 checkpoint pathway in mammals: linkageof DNA damage to Cdk regulation through Cdc25” Science 277:1497-1501)and Hcds1 (Matusoka et al., 1998, “Linkage of ATM to cell cycleregulation by the Chk2 protein kinase” Science 282(5395):1893-1897;Blasina et al., 1999, “A human homologue of the checkpoint kinase Cds1directly inhibits Cdc25 phosphatase” Curr. Biology 9(1):1-10).

Genetic and biochemical analysis of the checkpoint proteins in yeast andmammalian cells suggests that the checkpoint response is transmittedthrough a conventional signal transduction pathway. Hrad1, Hrad9,Hrad17, and Hhus1 transmit the signal emanating from damaged orincompletely replicated DNA to the central kinases ATM and ATR, which inturn activate the downstream kinases, Chk1 and Cds1. The DNA structurecheckpoint responses ultimately lead to phosphorylation of the mitosisinducing phosphatase Cdc25 by Chk1 or Cds1. This phosphorylation eventcreates a binding site for 14-3-3 proteins that target Cdc25 for exportfrom the nucleus to the cytoplasm, thus preventing it from removing aninhibitory phosphate from the cyclin dependent kinase, Cdc2. Removal ofthis inhibitory phosphate is required for passage from G2 to mitosis inevery cell cycle. The DNA structure checkpoint responses prevent thisfrom occurring and result in a G2/M arrest.

Whereas the Chk1 protein has been shown to be required for the G2/M DNAdamage checkpoint in S. pombe, the replication checkpoint requires theactivity of both Cds1 and Chk1. When replication is blocked by treatmentwith the ribonucleotide reductase inhibitor hydroxyurea (HU), wild typecells arrest prior to mitosis. A cds1chk1 double mutant fails to arrestin the presence of HU while both single mutants arrest normally(Russell, 1998, “Checkpoints on the road to mitosis” Trends inBiochemical Sciences 23(10):399-402). S. pombe Chk1 and Cds1 are bothcapable of phosphorylating Cdc25 and targeting it for binding by 14-3-3proteins. Activation of the S. pombe Cds1 protein kinase by HU alsoresults in enhanced binding to and phosphorylation of Wee1, andaccumulation of Mik1. These two protein kinases are required for theinhibitory phosphorylation of Cdc2 that prevents cells from enteringmitosis suggesting an alternative to Cdc25C phosphorylation forcheckpoint mediated cell cycle arrest. Recently, Cds1 has also beenshown to be required for a DNA damage checkpoint in S-phase (Rhind andRussell, 1998, “The Schizosaccharomyces pombe S-phase checkpointdifferentiates between different types of DNA damage” Genetics149(4):1729-1737; Lindsay et al., 1998, “S-phase-specific activation ofCds1 kinase defines a subpathway of the checkpoint response inSchizosaccharomyces pombe” Genes Dev. 12(3):382-395). A human homologueof S. pombe Cds1 that is activated by DNA damage and HU in anATM-dependent manner and is capable of phosphorylating Cdc25C in vitrowas recently identified (Matsuoka et al., 1998, supra; Blasina et al.,1999, supra). The human cDNA encodes a 543 amino acid protein which likeits S. pombe homologue, contains a forkhead associated (FHA) domainN-terminal to the kinase domain. FHA domains are found in several otherproteins including the S. cerevisiae Cds1 orthologue Rad53. Rad53contains two FHA domains, one of which is required for interaction withthe DNA damage checkpoint protein Rad9 in the presence of DNA damage(Sun et al., 1998, “Rad53 FHA domain associated with phosphorylated Rad9in the DNA damage checkpoint” Science 281(5374):272-274).

In order to develop new and more effective treatments and therapeuticsfor the amelioration of the effects of aging or disease such as cancer,it is important to identify and characterize mammalian, and inparticular human, checkpoint proteins and to identify mediators of theiractivity. The present invention teaches the identification andcharacterization of human and murine nucleic acids encoding human Mus81(Hmus81) and murine Mus81 (Mmus81) protein with significant homology tothe S. pombe Mus81 protein that interacts with the S. pombe Cds1 FHAdomain. The S. cerevisiae orthologue is reported to be involved inmeiosis and DNA repair.

As described below, a Hmus81 gene acts as a checkpoint/repair gene andis involved with DNA repair. The checkpoint/repair delays provide timefor repair of damaged DNA prior to its replication in S-phase and priorto segregation of chromatids in M-phase, and Hmus81 appears to act inboth aspects, similarly to other known checkpoint/repair genes. In manycases, the DNA-damage response pathways will cause arrest, and the cellwill fail to divide. However, a functional DNA repair mechanism willallow the damage to be corrected, and thus allow eventual cell divisionto occur.

In humans, excision repair is an important defense mechanism against twomajor carcinogens, sunlight and cigarette smoke. It has been found thatindividuals defective in excision repair exhibit a high incidence ofcancer. (see Sancar, A, 1996, “DNA Excision Repair” Ann. Rev. Biochem.65:43-81). Other mechanisms also act in a simiar manner to repair DNA,such as mismatch repair which stabilizes the cellular genome bycorrecting DNA replication errors and by blocking recombination eventsbetween divergent DNA sequences. Inactivation of genes encoding theseactivities results in a large increase in spontaneous mutability andpredisposition to tumor development. (see Modrich & Lahue, 1996,“Mismatch Repair in Replication Fidelity, Genetic Recombination andCancer Biology” Ann. Rev. Biochem. 65:101-33). The importance ofmaintaining fidelity in the DNA is amply illustrated by the manymechanisms for repair, and if unrepairable, arrest of cell division.(see Wood, R D, 1996, “DNA Repair in Eukaryotes” Ann. Rev. Biochem.65:135-67).

Many chemotherapeutic agents are designed to disrupt or otherwise causedamage to the DNA of the targeted malignant cells. Antineoplastic agentssuch as alkylating agents, antimetabolites, and other chemical analogsand substances typically act by inhibiting nucleotide biosynthesis orprotein synthesis, cross-linking DNA, or intercalating with DNA toinhibit replication or gene expression. Bleomycin and etoposide forexample, specifically damage DNA and prevent repair.

The inhibition of Hmus81 gene or protein activity amplifies the potencyof antineoplastic agents, and enhances the efficacy of their use aschemotherapeutic agents. This enhancement is beneficial in not only morethoroughly affecting the targeted cells, but by allowing for reduceddosages to be used in proportion to the increased efficacy, thusreducing unwanted side effects. Inhibition of Hmus81 or Mmus81 geneactivity via anti-sense nucleic acid pharmaceuticals can be effectedusing the nucleic acids of the invention as the template forconstructing the anti-sense nucleic acids. It is preferred to target theamino terminal end of the nucleic acid for anti-sense binding, and thusinhibition, as this reduces translation of the mRNA. Inhibition ofHmus81 protein activity can be effected by the use of altered orfragments of Hmus81 or Mmus81 protein to competitively inhibit thebiochemical cascade that results in the repair of damaged DNA, or tocause cell arrest.

Disease can also result from defective DNA repair mechanisms, andinclude hereditary nonpolyposis colorectal cancer (defect in mismatchrepair), Nijmegen breakage syndrome (defect in double strand breakrepair), Xeroderma pigmentosum, Cockayne syndrome, andTrocothiodystrophy (defect in nuclear excision repair). (see for exampleLengauer et al., 1998, “Genetic instabilities in human cancers” Nature396(6712):643-649; Kanaar et al., 1998, “Molecular mechanisms of DNAdouble stranded repair” Trends Cell Biol. 8(12):483-489).

It is further envisioned that the transient inhibition of Hmus81 gene orprotein activity can be sufficient to effect improved treatment of cellbehavior due to aging or disease. For example, the transient inhibitionof DNA checkpoint/DNA damage arrest of cell division may allow thecombined use of lower doses of chemotherapeutic agents to effect greaterdamage to targeted cells in the treatment of diseases such as cancer.

SUMMARY OF THE INVENTION

Novel genes and proteins encoded thereby are useful for modifying cellgrowth, division and death. One aspect of the invention is a novelmammalian, e.g., human or murine checkpoint/repair protein, the nucleicacids which encode for it and its protein variants, nucleic acidconstructs, and methods for the production and use of mammalian Mus81encoding gene and protein. As used herein, “checkpoint gene” means agene which encodes for a protein which acts in the checkpoint/repairregulation of cell division. Such protein can effect both replicationand DNA damage checkpoint activity, ie. having checkpoint/repairactivity. Specific characterization of the mammalian Mus81 proteinencoding nucleic acids and their role in cell cycle regulation providesfor novel and useful compounds for modulating the mammalian cell cyclein a target cell.

As used herein, the terms “human Mus81 gene”, “Hmus81 encoding gene” and“Hmus81 gene” encompas human Mus81 encoding genes, including the allelicvariants of the gene which will occur in a human population, but stillencode for the same protein, splice variants of the gene, as well as thetranscripts from such genomic genes, cDNA encoding for the transcript,and other nucleic acids which will encode a Hmus81 protein. As usedherein, the terms “human Mus81 protein”, “Hmus81” and “Hmus81 protein”refer generally to the protein expressed from a Hmus81 encoding nucleicacid, and includes splice variants and glycosylation variants of theprotein which are generated by the translation and processing of theprotein encoded by a Hmus81 encoding gene, and in particular to a humanMus81 protein having an amino acid sequence corresponding to thatdepicted as SEQ ID NO: 2, 4, 8, and 10. In a preferred embodiment, theisolated nucleic acids of the invention correspond to a cDNA thatencodes for a human Mus81 protein. Any particular isolated nucleic acidof the invention, preferably encodes for only one form of a human Mus81protein.

As described in detail below, the human Mus81 encoding nucleic acids ofthe invention encompasses isolated nucleic acids comprising a nucleotidesequence corresponding to the nucleotide sequences disclosed herein andspecifically identified as Human Mus81₁, (“Hmus81(1)”; SEQ ID NO: 1),Human Mus81₂ (“Hmus81(2)”; SEQ ID NO: 3), Human Mus81₃ (“Hmus81(3)”; SEQID NO: 7), and Human Mus81₄ (“Hmus81(4)”; SEQ ID NO: 9). All of theforegoing nucleic acids encode for a human Mus81 protein, and itsequivalents. Thus, the present invention encompasses a nucleic acidhaving a nucleotide sequence which encodes for a Hmus81 protein andspecifically encompasses a nucleotide sequence corresponding to thecoding domain segment of the sequences that are depicted as SEQ ID NO:1, 3, 7, 9 and 25.

The present invention also encompasses a nucleic acid which encodes fortwo versions of Hmus81 protein having a nucleotide sequencecorresponding to that depicted as SEQ ID NO:25. This nucleic acidencodes for a Hmus81 protein having an amino acid residue sequencedepicted as SEQ ID NO: 4, wherein the 201 nucleotides from position 1274to 1474 of the sequence of SEQ ID NO: 25 containing a stop codon, havebeen deleted, thus allowing translation of the longer coding domainsegment sequence of DNA. The nucleic acid having a correspondingnucleotide sequence as that depicted as SEQ ID NO: 25 also encodes forthe shorter Hmus81 protein having the amino acid sequence depicted asSEQ ID NO: 2, from a shorter coding domain segment, leaving the intronin place.

Thus, in a preferred embodiment, the present invention encompassesnucleic acids which encode for human Mus81 proteins, and in particular,nucleic acids having a coding domain segment sequence corresponding tothat represented by nucleotides 23-1675 of the nucleotide sequencedepicted as SEQ ID NO: 1; to that represented by nucleotides 185-1549 ofthe nucleotide sequence depicted as SEQ ID NO:3; to that represented bynucleotides 26-1297 of the nucleotide sequence depicted as SEQ ID NO:7;to that represented by nucleotides 26-1681 of the nucleotide sequencedepicted as SEQ ID NO:9; or as identified in SEQ ID NO: 25.

The terms “murine Mus81 gene” and “Mmus81 gene” are used herein to referto the novel murine Mus81 encoding genes. The terms “murine Mus81protein”, “Mmus81” and “Mmus81 protein” refer generally to the proteinproduct of the Mmus81 genes and in particular, to murine Mus81 proteinshaving an amino acid residue sequence corresponding to that depicted asSEQ ID NO: 12, 14, 16, and 18.

The terms “murine Mus81 gene”, “Mmus81 gene” and “Mmus81 encoding gene”encompass the Mmus81 genes, and in particular isolated nucleic acidscomprising a nucleotide sequence corresponding to the nucleotidesequences disclosed herein and identified as Mouse (murine) Mus81₁,(“Mmus81(1)”; SEQ ID NO: 11), Mouse Mus81₂ (“Mmus81(2)”; SEQ ID NO: 13),Mouse Mus81₃ (“Mmus81(3)”; SEQ ID NO: 15), and Mouse Mus81₄(“Mmus81(4)”; SEQ ID NO: 17), and the protein coding domain segmentsencoded for therein. In a preferred embodiment, the isolated nucleicacids of the invention correspond to a cDNA that encodes for a murineMus81 protein. Any particular isolated nucleic acid of the invention,preferably encodes for only one form of a murine Mus81 protein.

In another preferred embodiment, the present invention encompassesnucleic acids which encode for murine Mus81 proteins, and in particular,nucleic acids which have a coding domain segment sequence correspondingto that represented by nucleotides 42-1694 of the nucleotide sequencedepicted as SEQ ID NO: 11; to that represented by nucleotides 15-1323 ofthe nucleotide sequence depicted as SEQ ID NO:13; to that represented bynucleotides 52-1644 of the nucleotide sequence depicted as SEQ ID NO:15;or to that represented by nucleotides 52-1614 of the nucleotide sequencedepicted as SEQ ID NO: 17.

The present invention also encompasses nucleic acid constructs, vectors,plasmids, cosmids, retrovirus or viral constructs and the like whichcontain a nucleotide sequence encoding for a human Mus81 or murine Mus81protein. In particular, the present invention provides for nucleic acidvector constructs which contain the nucleotide sequence of the Hmus81coding domain segments of the nucleic acid depicted as SEQ ID NO: 1, 3,7, 9 or 25 and which are expressible as a protein. The present inventionalso provides for nucleic acid vector constructs which contain theMmus81 coding domain segments of the nucleic acids depicted as SEQ IDNO: 11, 13, 15, or 17.

The term “transgene capable of expression” as used herein means asuitable nucleotide sequence which leads to expression of Hmus81 orMmus81 proteins, having the same function and/or the same or similarbiological activity as such protein. The transgene can include, forexample, genomic nucleic acid isolated from mammalian cells (e.g. humanor mouse) or synthetic nucleic acid, including DNA integrated into thegenome or in an extrachromosomal state. Preferably, the transgenecomprises the nucleotide sequence encoding the proteins according to theinvention as described herein, or a biologically active portion of saidprotein. A biologically active protein should be taken to mean, and notlimited to, a fusion product, fragment, digestion fragment, segment,domain or the like of a Mus81 protein having some if not all of theprotein activity as a whole Mus81 protein. A biologically active proteinthus contains a biologically functional portion of a mammalian Mus81protein conveying a biochemical function thereof.

The present invention encompasses nucleic acid vectors that are suitablefor the transformation of host cells, whether eukaryotic or prokaryotic,suitable for incorporation into viral vectors, or suitable for in vivoor in vitro protein expression. Particularly preferred host cells forprokaryotic expression of protein include, and are not limited tobacterial cells such as E. coli. Suitable host cells for eukaryoticexpression of protein include, and are not limited to mammalian cells ofhuman or murine origin and the like, or yeast cells. In a preferredembodiment, expression of protein, as described below, is accomplishedby viral vector transformation of immortalized human cells.

The present invention further embodies a nucleotide sequence whichencodes for a human Mus81 or murine Mus81 protein, in tandem with, orotherwise in conjunction with additional nucleic acids for thegeneration of fusion protein products. Human Mus81 fusion proteins willcontain at least one segment of the protein encoded for by the nucleicacid depicted as the coding domain segment depicted in the nucleotidesequence described as SEQ ID NO: 1, 3, 7, and 9. Similarly, murine Mus81fusion protein will contain at least one segment of protein encoded forby the coding domain segments of the nucleic acid depicted as SEQ ID NO:11, 13, 15, and 17.

The present invention also encompasses isolated nucleic acids or nucleicacid vector constructs containing nucleic acid segments, adapted for useas naked DNA transformant vectors for incorporation and expression intarget cells. Also provided are inhibitors of human Mus81 or murineMus81 encoding nucleic acid transcripts, such as anti-sense DNA,triple-helix nucleic acid, double-helix RNA or the like. Biologicallyactive anti-sense DNA molecule formulations are those which are thecomplement to the nucleotide sequence of the human Mus81 or murine Mus81encoding genes or fragments thereof, whether complementary to contiguousor discontinuous portions of the targeted nucleotide sequence, and areinhibitors of the human Mus81 or murine Mus81 protein expression incells. Such inhibitors and inhibition are useful for many purposesincluding and not limited to, in vitro analysis of the cell-cyclecheckpoint pathway, detection and/or evaluation of inhibiting orpotentiating compounds, and for in vivo therapy.

The present invention also provides for compositions incorporatingmodified nucleotides or substitute backbone components which encode forthe nucleotide sequence of a human Mus81 or murine Mus81 encoding gene,or fragments thereof.

The present invention encompasses the use of anti-sense nucleic acidswhich comprise a nucleic acid that is the complement of at least aportion of a nucleic acid encoding for a human Mus81 or murine Mus81protein. Also envisioned are biologically active analogs of thisantisense molecule selected from the group consisting of peptide nucleicacids, methylphosphonates and 2-O-methyl ribonucleic acids. An antisensemolecule of the invention can also be a phosphorothioate analog.

Also encompassed are pharmaceutical preparations for inhibiting Hmus81protein expression or function in a cell which comprises an antisensenucleic acid analog which is capable of entering said cell and bindingspecifically to a nucleic acid molecule encoding for Hmus81 protein. Theantisense nucleic acid is present in a pharmaceutically acceptablecarrier and has a nucleotide sequence complementary to at least aportion of the nucleic acid of SEQ ID NO: 1, 3, 7, 9 or 25. It is alsoenvisioned that this pharmaceutical preparation can comprise a nucleicacid having a sequence complementary to at least the nucleotidesencoding for amino acid residues 1-50 of the amino acid residue sequenceof SEQ ID NO: 2, 4, 8, or 10. In a preferred embodiment, thepharmaceutical preparation comprises a nucleic acid having a nucleotidesequence complementary to at least nucleotides 1-20 of a coding domainsegment in the nucleotide sequence depicted as SEQ ID NO: 1, 3, 7, 9 or25. In a most preferred embodiment, the antisense nucleic acid comprisesa nucleic acid having a sequence complementary to at least nucleotides1-10 of a coding domain segment in the nucleotide sequence depicted asSEQ ID NO: 1, 3, 7, 9 or 25.

The present invention also encompasses nucleotide sequences which wouldencode for the Hmus81 protein having an amino acid sequence as thatdepicted by that of SEQ ID NO: 2, 4, 8 or 10 based upon synonymous codonsubstitution given the knowledge of the triplet codons and which aminoacids they encode, based upon the coding domain segment of thenucleotide sequence depicted in SEQ ID NO. 1, 3, 7, 9 or 25. Theequivalent synonymous nucleic acid code for generating any nucleotidesequence which will encode for a protein having a particular amino acidsequence is known and predictable to one of skill in the art.

In a preferred embodiment codon usage is optimized to increase proteinexpression as desired for the target host cell, such as where a nucleicacid is modified so that it comprises a protein coding domain segment ofthe nucleotide sequence depicted in SEQ ID NO: 1, 3, 7, 9, 11, 13, 15,17 or 25, wherein the least preferred codons are substituted with thosethat are most preferred in the target host cell. In the case of humantarget host cells, the least preferred codons are ggg, att, ctc, tcc,and gtc.

The invention also provides for methods of generating human Mus81 ormurine Mus81 protein, fusion proteins, or fragments thereof by usingrecombinant DNA technology and the appropriate nucleic acid encoding forhuman Mus81 or murine Mus81 protein. The invention provides forincorporating an appropriate nucleotide sequence into a suitableexpression vector, the incorporation of suitable control elements suchas a ribosome binding site, promoter, and/or enhancer element, eitherinducible or constitutively expressed. The invention provides for theuse of expression vectors with or without at least one additionalselectable marker or expressible protein. The invention provides formethods wherein a suitably constructed expression vector is transformedor otherwise introduced into a suitable host cell, and protein isexpressed by such a host cell. The present invention also providestransformed host cells, which are capable of producing human Mus81 ormurine Mus81 protein, fusion protein, or fragments thereof. Theexpression vector including said nucleic acid according to the inventionmay advantageously be used in vivo, such as in, for example, genetherapy.

The invention encompasses mammalian, e.g. human or murine Mus81 protein,fusion products, and biologically active portions thereof produced byrecombinant DNA technology and expressed in vivo or in vitro. Abiologically active portion of a protein is protein segment or fragmenthaving the enzymatic activity of, or at least a some enzymatic activityof the whole mammalian Mus81 protein, when compared under similarconditions. For example, it will be readily apparent to persons skilledin the art that nucleotide substitutions or deletions may be introducedusing routine techniques, which do not affect the protein sequenceencoded by said nucleic acid, or which encode a biologically active,functional protein according to the invention. Manipulation of theprotein to generate fragments as a result of enzyme digestion, or themodification of nucleic acids encoding for the protein can similarlyresult in biologically active portions of the mammalian Mus81 protein.

Complete protein, fusion products and biologically active portionsthereof of the mammalian Mus81 protein are useful for therapeuticformulations, diagnostic testing, and as immunogens, as for example togenerate antibodies thereto. The invention thus encompasses Hmus81 andMmus81 protein produced by transformed host cells in small-scale orlarge-scale production. The invention encompasses complete Hmus81 andMmus81 protein, in either glycosylated or unglycosylated forms, producedby either eukaryotic or prokaryotic cells. The present inventionprovides for Hmus81 and Mmus81 protein expressed from mammalian, insect,plant, bacterial, fungal, or any other suitable host cell using theappropriate transformation vector as known in the art. The presentinvention encompasses Hmus81 and Mmus81 protein that is produced as afusion protein product, conjugated to a solid support, or Hmus81 andMmus81 protein which is labeled with any chemical, radioactive,fluorescent, chemiluminescent or otherwise detectable marker.

The present invention also provides Hmus81 and Mmus81 proteins isolatedfrom natural sources and enriched in purity over that found in nature.Also provided are pharmaceutical formulations of Hmus81 and Mmus81protein as well as formulations of the Hmus81 and Mmus81 protein inpharmaceutically acceptable carriers or excipients.

The present invention also encompasses the use of human Mus81 or murineMus81 protein, fusion protein, or biologically active fragments thereofto generate specific antibodies which bind specifically to the humanMus81 or murine Mus81 protein, or both, as either polyclonal ormonoclonal antibodies generated by the immunization of a mammal withhuman Mus81 protein having the amino acid residue sequence, or animmunogenic fragment of the amino acid residue sequence shown as SEQ IDNO: 2, 4, 8, or 10, or the murine Mus81 protein having the amino acidresidue sequence shown as SEQ ID NO: 12, 14, 16 or 18. An immunogenicfragment is one which will elicit an immune response, when injected intoa immunologically competent host under immunogenic conditions, andgenerate antibodies specific for the immunogenic fragment.

The present invention also encompasses equivalent proteins wheresubstitutions of amino acids for amino acid residues as shown in theamino acid sequence encoding for human Mus81 protein (SEQ ID NO: 2, 4,8, 10) or murine Mus81 protein (SEQ ID NO:12, 14, 16, 18) are made. Suchamino acid substitutions include conservative substitutions of similaramino acid residues that are reasonably predictable as being equivalent,or semi-conservative substitutions which have a reasonably predictableeffect on solubility, glycosylation, or protein expression. For example,non-polar (hydrophobic side-chain) amino acids alanine, valine, leucine,isoleucine, proline, phenylalanine, tryptophan, methionine; unchargedpolar amino acids glycine, serine, threonine, cysteine, tyrosine,asparagine, glutamine; charged polar amino acids aspartic acid, glutamicacid; basic amino acids lysine, arginine, and histidine are understoodby those in the art to have functionally predictable effects whensubstituted. Amino acid substitutions also include replacement of aminoacid residues with modified amino acid residues or chemically alteredsubstitutes.

The present invention also encompasses nucleic acids which encode forsuch equivalent proteins and the embodiments thereof which encode forthe human Mus81 proteins or murine Mus81 proteins. Specific modificationcan be made of codons used in the nucleic acids corresponding to thehuman Mus81 or murine Mus81 encoding genes of the invention such thatthe modified nucleic acids utilize codons preferred by the target hostcell, while still encoding for the human Mus81 or murine Mus81 protein.This can be accomplished by conservative synonymous codon substitutionsthat reduce the number of less preferred codons and/or an increase inthe number of preferred codons used by the target host cell The presentinvention also encompasses modified nucleic acids which incorporate, forexample, internucleotide linkage modification, base modifications, sugarmodification, nonradioactive labels, nucleic acid cross-linking, andaltered backbones including PNAs (polypeptide nucleic acids).

The knowledge that Hmus81 acts as a checkpoint/repair protein and ismost likely involved in DNA repair, allows for the use of the compoundsof the invention in therapeutic treatment of diseases which involveabnormal DNA damage checkpoint/repair function, or that wouldadvantageously inhibit DNA repair in a targeted cell. The presentinvention further provides for the use of the compounds of the presentinvention as therapeutics for the treatment of cancer. In oneembodiment, inhibitors or agents which inhibit the function of thenormal proteins and/or genes of the invention would be useful tosensitize cells for treatment with chemotherapeutics, radiation, DNAdamaging agents, or replication inhibitors.

The present invention also encompasses methods for screening testcompounds for efficacy in effecting the Mus81 mediated checkpoint/repairfunction of eukaryotic cells. These methods comprise contacting a testcompound to eukaryotic cells, and detecting any change in mammalianMus81 expression or function. Also encompassed are methods of screeningwherein a compound is administered, and detection of change in Hmus81 orMmus81 gene expression or function is accomplished by assaying forHmus81 or Mmus81 mRNA production or by assaying for Hmus81 or Mmus81protein expression. Methods for detection of changes in expression levelof a particular gene are known in the art. In particular, the presentinvention allows for the screening of candidate substances for efficacyin modifying the mammalian Mus81 mediated DNA damage checkpoint/repairor DNA repair function by screening for any change in nuclease,phosphorylation or kinase activity of mammalian Mus81 protein. Thecompounds or substances identified by the assays of the invention, orcompounds corresponding to such compounds or substances, can be used forthe manufacture of pharmaceutical therapeutics.

Methods of identifying a chemical compound that modulates the Mus81dependent cell cycle pathway are provided for as well. Such methodscomprise administering the chemical compound to be tested to a hostcell, and detecting the amount of mammalian Mus81 protein in said cell,and comparing the amount detected with that of a normal untreated cell.Further provided for is a method of identifying a chemical compound thatmodulates the Mus81 dependent cell cycle pathway, which method comprisesadministering the chemical compound to be tested to a biochemicalmixture of Hmus81 protein and a suitable substrate, and detecting thelevel of Hmus81 protein activity in said mixture, and comparing thedetected activity with that of a normal untreated biochemical mixture ofHmus81 protein. As shown in the examples below, isolated Hmus81 proteinand suitable substrates can be measured in isolated chemical reactions.

In one embodiment, the present invention also provides forpharmaceutical compositions which comprise the Hmus81 protein, Hmus81nucleic acid, or Hmus81 anti-sense nucleic acids. The therapeutic Hmus81protein can be normally glycosylated, modified, or unglycosylateddepending upon the desired characteristics for the protein. Similarly,Hmus81 protein includes the complete long or short protein, fusionproduct, or functional or immunogenic fragment thereof. Hmus81 nucleicacids include those encoding for the entire long or short protein,portions of the protein, fusion protein products, and fragments thereof.Also included are modified forms of nucleic acids including thoseincorporating substitute base analogs, modified bases, PNAs and thoseincorporating preferred codon usage. Anti-sense nucleic acids includecomplementary nucleic acids which can bind specifically to the targetednucleic acids, having full, part or discontinuous segments ofcomplementary nucleic acid which can be DNA, RNA or analog compoundsthereof. In another embodiment, the present invention provides forcompounds or substances identified as suitable for use as a therapeuticin pharmaceutical formulations by the assays of the invention. Thesepharmaceutical compositions can further include chemotherapeutic agentsfor the use in treating cancer, or be administered in a regimencoordinated with the administration of other anti-cancer therapies. Thepresent invention, in one embodiment, encompasses methods for combinedchemotherapy using the Hmus81 derived pharmaceuticals independently, andin combination with other chemotherapeutic agents, and in a secondembodiment as admixtures with other anti-cancer therapeutics for singledose administration.

Similarly, murine Mus81 protein, or nucleic acids encoding for theprotein can be used to modulate the cell cycle of murine or non-murinemammalian cells. Nucleic acids encoding for the murine Mus81 protein,can be used to produce murine Mus81 protein by recombinant means for usein pharmaceuticals, detection methods and kits, and assay systems in thesame manner as human Mus81 protein.

The invention provides for a transgenic cell, transformed cell, tissueor organism comprising a transgene capable of expressing human Mus81protein, which protein comprises the amino acid sequence illustrated inFIG. 1A (SEQ ID NO:2), FIG. 1B (SEQ ID NO:4), FIG. 1C (SEQ ID NO:8),FIG. 1D (SEQ ID NO:10), or a murine Mus81 protein, which proteincomprises the amino acid sequence illustrated in FIG. 2A (SEQ ID NO:12),FIG. 2B (SEQ ID NO:14), FIG. 2C (SEQ ID NO:16), FIG. 2D (SEQ ID NO: 18),or the amino acid sequence of a biologically active functionalequivalent or bioprecursor or biologically active fragment therefor. Andfor the isolated protein produced by such transformed host cells.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention may be better understood by reference to one or more ofthe following drawings in combination with the detailed description ofspecific embodiments, and claims presented herein.

FIG. 1 depicts nucleotide sequences of human Mus81 cDNA molecules andamino acid sequences of their translation products. FIG. 1A depicts thenucleotide sequence of a PCR product from cerebellum cDNA libraryencoding a 551 amino acid protein Hmus81(1) (SEQ ID NO: 1 and 2). FIG.1B depicts the nucleotide sequence of IMAGE 128349 cDNA encoding a 455amino acid protein Hmus81(2) (SEQ ID NO: 3 and 4). FIG. 1C depictsSequence of a PCR product from cerebellum cDNA library encoding a 424amino acid protein Hmus81(3) (SEQ ID NO: 7 and 8). FIG. 1D depicts anucleotide sequence of a PCR product from cerebellum cDNA libraryencoding a 552 amino acid protein Hmus81(4) (SEQ ID NO: 9 and 10).

FIG. 2 depicts nucleic acid nucleotide sequences of mouse Mus81 cDNAmolecules and amino acid sequences of their translation products. FIG.2A depicts a nucleic acid encoding for Mmus81(1) and the amino acidsequence for the translated protein of 551 amino acids in length (SEQ IDNO: 11 and 12). FIG. 2B depicts a nucleic acid encoding for Mmus81(2)and the amino acid sequence for the translated protein of 424 aminoacids in length (SEQ ID NO: 13 and 14). FIG. 2C depicts a nucleic acidencoding for Mmus81(3) and the amino acid sequence for the translatedprotein of 531 amino acids in length (SEQ ID NO: 15 and 16). FIG. 2Ddepicts a nucleic acid encoding for Mmus81(4) and the amino acidsequence for the translated protein of 521 amino acids in length (SEQ IDNO: 17 and 18).

FIG. 3 graphically presents an alignment of mouse (Mm) (Mmus81; SEQ IDNO:12), human (Hs) (Hmus81; SEQ ID NO:10), S. pombe (Sp) (Spmus81; SEQID NO:6), and S. cerevisiae (Sc) Mus81 (Scmus81; SEQ ID NO:5) amino acidsequences. Amino acids conserved in all proteins are highlighted inblack and in two or more proteins in grey. Sequences underlined in redcorrespond to the conserved catalytic domain of the XPF family ofendonucleases.

FIG. 4. Genomic structure and splicing variations of human Mus81. Solidline represents genomic sequence and boxes indicate positions of exons.Sizes of exons and introns (in bp) are indicated above and below thegenomic fragment, respectively. Alternative splicing that occurs aroundexons 13 and 14 corresponds to Mus81₁, Mus81₄, and Mus81₃, is shown bythin lines. Mus81₂ utilizes all the identified exons.

FIG. 5. Chromosomal localization of human Mus81 by FISH analysis. (A)Chromosome metaphase spread labelled with a fluorescent Mus81 cDNA probe(left panel) and corresponding DAPI staining (right panel). (B) Idiogramof chromosome 11 with location of Hybridisation signal from 10representative metaphase spreads.

FIG. 6. Northern blot analysis of human Mus81. Human tissues (H1 and H2)and cancer cell lines (C).

FIG. 7. Cellular localization of a Mus81-GFP (GFP: Green FluorescentProtein e.g. from Aequorea Victoria) fusion protein. A549 cells infectedwith a retrovirus expressing a Hmus81-GFP fusion at 3 days afterinduction.

FIG. 8. Co-immunoprecipitation of human Mus81 and Cds1. Western blots oflysates (L) and immunoprecipitates (IP) from cells expressing taggedforms of Mus81 and Cds1 separately and together. Bands corresponding toMus81 and Cds1 are indicated with arrows. Bands corresponding to aprotein that cross-reacts with the HA antibody in the upper panelindicated by an asterisk. Immunoglobulin heavy chains in the lower panelare indicated by an arrowhead.

DETAILED DESCRIPTION OF THE INVENTION

The present invention, in one aspect, provides for isolated nucleicacids which encode for novel mammalian cell cycle check-point/repairproteins, such as human Mus81 proteins and murine Mus81 proteins and thelike. The nucleic acids of the invention are useful for generating humanMus81 or murine Mus81 proteins using recombinant DNA techniques, fortransforming target host cells as naked nucleic acid vectors, or whenconstructed in combination with nucleic acid regulatory elements such aspromoters, enhancers, or supressors as expression vector constructs.Advantageously, the nucleic acid molecules according to the inventioncan be used as a medicament, or in the preparation of a medicament formodulating cell cycle checkpoint/repair functions of a target cell, forthe treatment of cancer and other proliferative diseases.

The present invention also provides for isolated and/or recombinantlyproduced human and murine Mus81 proteins, and protein analogs.Recombinantly produced human Mus81 or murine Mus81 proteins of theinvention can be used advantageously in vitro or in vivo for modulatingthe cell cycle and/or checkpoint/repair pathway of a targeted host cell.Isolated human Mus81 or murine Mus81 protein of the present inventionmay be utilized to generate antibodies which bind specifically to thehuman Mus81 protein and/or murine Mus81 protein, where such antibodiescan be either polyclonal or monoclonal. Advantageously, the proteinmolecules according to the invention can be used as a medicament, or inthe preparation of a medicament for modulating cell cyclecheckpoint/repair functions of a target cell, for the treatment ofcancer and other proliferative diseases.

Isolated recombinantly produced human and/or murine Mus81 proteins canalso be used in combination with other proteins as in vitro biochemicalsystems for modeling enzymatic steps of an in vivo cell cyclecheckpoint/repair pathway for testing and/or evaluating chemical orprotein compounds for the ability to modulate the cell cyclecheckpoint/repair mechanism associated with human Mus81 or murine Mus81protein. A biochemical mixture of human Mus81 or murine Mus81 proteinwill comprise the isolated enzyme, appropriate ions and/or cofactors,and suitable substrate. A preferred biochemical mixture will comprise asuitable substrate which will detectably change or signal a change instate, when the enzymatic activity of the Mus81 protein has been appliedto the substrate, for example by emission of energy, or fluorescentlight, or an alteration in the wavelength of emitted light energy, or bya change in binding by a antibody molecule specific for a particularform of Mus81 protein.

The isolated nucleic acids of the invention, and the nucleotide sequenceencoded by them, provide for isolated DNA, RNA, modified nucleotideanalog, or labeled nucleic acid constructs which can mimic,complementarily bind to, and/or otherwise label nucleic acids comprisingthe same or highly related nucleotide sequences in nucleic acids invitro or in vivo. It is envisioned that the nucleic acids of theinvention can incorporate modified nucleotides and nucleic acid baseanalogs, which are known in the art (see for example Verma et al., 1998,“Modified Oligonucleotides” Ann. Rev. Biochem. 67: 99-134). The isolatednucleic acids of the present invention can be a biologically activeantisense molecule, which is one capable of hybridizing to a targetnucleic acid upon the complementary binding of nucleic acids and therebymodulate the expression of the targeted nucleic acid. Advantageously,the antisense molecule according to the invention can be used as amedicament, or in the preparation of a medicament for modulating cellcycle checkpoint/repair functions of a target cell, for the treatment ofcancer and other proliferative diseases. Suitable biologically activeantisense nucleic acids comprise modified nucleotide bases or the likefor improving the stabilization of such nucleic acids or resistance tonucleases, such as (2′-O-(2-methoxy)ethyl (2′-MOE) modification ofoligonucleotides (McKay et al., 1999, “Characterization of a potent andspecific class of antisense oligonucleotide inhibitors of humanPKC-alpha expression” J. Biol. Chem. 274:1715-1722). Preferred antisensenucleic acid molecules are at least 10 residues in length, preferably 20residues in length, and are directed to a portion of the gene transcriptthat will result in the inhibition of translation of a functionalprotein from the gene transcript.

The present invention also advantageously provides for nucleotidesequences of at least approximately 15 nucleotides which arecomplementary to a contiguous portion of a nucleic acid according to theinvention. These complementary sequences can be used as probes orprimers to initiate replication, to detect the presence of nucleic acidshaving the nucleotide sequence of the invention, or to specificallyamplify segments of the desired nucleic acid from a sample. Suchcomplementary nucleotide sequences can be produced according totechniques well known in the art, such as by recombinant or syntheticmeans. The prepared primers, properly coordinated to specificallyamplify a portion of a target nucleic acid in a sample may be used indiagnostic kits, or the like, for detecting the presence of a nucleicacid according to the invention. These tests generally comprisecontacting the probe with the sample under hybridizing conditions anddetecting for the presence of any duplex or triplex formation betweenthe probe and any nucleic acid in the sample.

Advantageously, the nucleotide sequences embodying the invention can beproduced using such recombinant or synthetic means, such as for exampleusing PCR cloning mechanisms which generally involve making a pair ofprimers, which may be from approximately 15 to 50 nucleotides to aregion of the gene which is desired to be cloned, bringing the primersinto contact with mRNA, cDNA, or genomic DNA from a human cell,performing a polymerase chain reaction under conditions which bringabout amplification of the desired region (and where necessary firstperforming a reverse transcription step), isolating the amplified regionor fragment and recovering the amplified DNA. Advantageously, humanallelic variants of the nucleic acid according to the invention can beobtained by for example, probing genomic DNA libraries from a range ofindividuals for example from different populations, and other genotypingtechniques. Furthermore, nucleic acids and probes according to theinvention may be used to sequence genomic DNA from patients, usingtechniques well known in the art, for example, the Sanger dideoxy chaintermination method, which can advantageously ascertain anypredisposition of a patient to certain proliferative disorders.

Specific modification of codons used in the nucleic acids correspondingto SEQ ID NO: 1, 3, 7, and 9 can be such that the modified nucleic acidsutilize codons preferred by the target host cell, while still encodingfor the Hmus81 protein. Similarly, the present invention encompassesspecific modification of codons used in the nucleic acids correspondingto SEQ ID NO: 11, 13, 15, and 17 such that the modified nucleic acidsutilize codons preferred by the target host cell, while still encodingfor the Mmus81 protein. The present invention also encompasses modifiednucleic acids which incorporate, for example, internucleotide linkagemodification, base modifications, sugar modification, nonradioactivelabels, nucleic acid cross-linking, and altered backbones including PNAs(polypeptide nucleic acids), as well as codon substitutions to reducethe number of less preferred codons and/or an increase in the number ofpreferred codons used by the target host cell (see Zhang et al., 1991,“Graphic analysis of codon usage strategy in 1490 human proteins” Gene105(1):61-72; Zhang et al., 1993, “Low-usage codons in Escherichia coli,yeast, fruit fly and primates” J. Protein Chemistry 12(3):329-335).

According to one aspect of the present invention, there is provided anucleic acid encoding Hmus81 protein having the amino acid residuesequence as illustrated as SEQ ID NO: 2, 4, 8, or 10, or encoding afunctionally equivalent fragment, or bioprecursor of said protein.According to another aspect of the present invention, there is provideda nucleic acid encoding Mmus81 protein having the amino acid residuesequence as illustrated as SEQ ID NO: 12, 14, 16, or 18, or encoding afunctionally equivalent fragment, or bioprecursor of said protein.

Preferably, the nucleic acid is a DNA molecule such as a genomic DNAmolecule, and even more preferably a cDNA molecule. However, it may alsobe RNA. As is well known to those skilled in the art, due to thedegeneracy of the triplet codon genetic code, the present nucleotidesequences can include substitutions therein yet which still encode thesame amino acid residue sequence.

The nucleotide sequences defined herein are capable of hybridizing underlow stringency conditions to nucleotide sequences derived from a nucleicacid of the invention, to identify homologs therefrom or alternativelyto identify nucleotide sequences from other species.

The present nucleic acids can be incorporated into an expression vectorand subsequently used to transform, transfect or infect a suitable hostcell. In such an expression vector the nucleic acid according to theinvention is operably linked to a control sequence, such as a suitablepromoter or the like, ensuring expression of the proteins according tothe invention in a suitable host cell. The expression vector can be aplasmid, cosmid, virus or other suitable vector. The expression vectorand the host cell transfected, transformed or infected with the vectoralso form part of the present invention. Preferably, the host cell is aeukaryotic cell or a bacterial cell and even more preferably a mammaliancell or insect cell. Mammalian host cells are particularly advantageousbecause they provide the necessary post-translational modifications tothe expressed proteins according to the invention, such as glycosylationor the like, which modifications confer optimal biological activity ofsaid proteins, which when isolated can advantageously be used indiagnostic kits or the like.

The recombinant vectors of the invention generally comprise a Hmus81gene or Mmus81 operatively positioned downstream from a promoter. Thepromoter is capable of directing expression of the human Mus81 or murineMus81 encoding nucleic acid in a mammalian, e.g. human cell. Suchpromoters are thus “operative” in mammalian cells, e.g. human cells.

Expression vectors and plasmids embodying the present invention compriseone or more constitutive promoters, such as viral promoters or promotersfrom mammalian genes that are generally active in promotingtranscription. Examples of constitutive viral promoters include the HSV,TK, RSV, SV40 and CMV promoters, of which the CMV promoter is acurrently preferred example. Examples of constitutive mammalianpromoters include various housekeeping gene promoters, as exemplified bythe β-actin promoter.

Inducible promoters and/or regulatory elements are also contemplated foruse with the expression vectors of the invention. Examples of suitableinducible promoters include promoters from genes such as cytochrome P450genes, heat shock protein genes, metallothionein genes,hormone-inducible genes, such as the estrogen gene promoter, and suchlike. Promoters that are activated in response to exposure to ionizingradiation, such as fos, jun and erg-1, are also contemplated. ThetetVP16 promoter that is responsive to tetracycline is a currentlypreferred example.

Tissue-specific promoters and/or regulatory elements will be useful incertain embodiments. Examples of such promoters that can be used withthe expression vectors of the invention include promoters from the liverfatty acid binding (FAB) protein gene, specific for colon epithelialcells; the insulin gene, specific for pancreatic cells; thetransphyretin, α1-antitrypsin, plasminogen activator inhibitor type 1(PAI-1), apolipoprotein AI and LDL receptor genes, specific for livercells; the myelin basic protein (MBP) gene, specific foroligodendrocytes; the glial fibrillary acidic protein (GFAP) gene,specific for glial cells; OPSIN, specific for targeting to the eye; andthe neural-specific enolase (NSE) promoter that is specific for nervecells.

The construction and use of expression vectors and plasmids is wellknown to those of skill in the art. Virtually any mammalian cellexpression vector can thus be used in connection with the genesdisclosed herein.

Preferred vectors and plasmids are constructed with at least onemultiple cloning site. In certain embodiments, the expression vectorwill comprise a multiple cloning site that is operatively positionedbetween a promoter and a human Mus81 or murine Mus81 encoding genesequence. Such vectors can be used, in addition to uses in otherembodiments, to create N-terminal or C-terminal fusion proteins bycloning a second protein-encoding DNA segment into the multiple cloningsite so that it is contiguous and in-frame with the mammalian Mus81encoding nucleotide sequence.

In other embodiments, expression vectors comprise a multiple cloningsite that is operatively positioned downstream from the expressiblehuman Mus81 or murine Mus81 encoding sequence. These vectors are useful,in addition to their uses, in creating C-terminal fusion proteins bycloning a second protein-encoding DNA segment into the multiple cloningsite so that it is contiguous and in-frame with the human Mus81 ormurine Mus81 encoding sequence.

Vectors and plasmids in which additional protein- or RNA-encodingnucleic acid segment(s) is(are) also present are, of course, alsoencompassed by the invention, irrespective of the nature of the nucleicacid segment itself.

A second reporter gene can be included within an expression vector ofthe present invention. The second reporter gene can be comprised withina second transcriptional unit. Suitable second reporter genes includethose that confer resistance to agents such as neomycin, hygromycin,puromycin, zeocin, mycophenolic acid, histidinol and methotrexate.

Expression vectors can also contain other nucleotide sequences, such asIRES elements, polyadenylation signals, splice donor/splice acceptorsignals, and the like.

Particular examples of suitable expression vectors are those adapted forexpression using a recombinant adenoviral, recombinant adeno-associatedviral (AAV) recombinant retroviral system. Vaccinia virus, herpessimplex virus, cytomegalovirus, and defective hepatitis B viruses,amongst others, can also be used.

In one specific embodiment, the present invention encompasses isolatednucleic acids which encode for novel mammalian checkpoint/repairproteins. In another specific embodiment, the invention encompassesnovel mammalian checkpoint/repair proteins derived from nucleic acidsisolated from a human source called Hmus81 (human Mus81), and from amurine source called Mmus81 (murine Mus81).

Further provided by the present invention are isolated proteins havingan amino acid residue sequence corresponding to that illustrated as SEQID NO: 2, 4, 8 or 10, or the amino acid sequence of a functionallyequivalent fusion protein product, fragment or bioprecursor of saidprotein. Also provided by the present invention are isolated proteinshaving an amino acid sequence corresponding to that illustrated as SEQID NO: 12, 14 16 or 18, or the amino acid residue sequence of afunctionally equivalent, fusion protein product, biologically activefragment or bioprecursor of said protein. Also envisioned is the use ofsuch protein for the generation of antibodies, monoclonal or polyclonalcapable of specifically binding to the amino acid sequences of theseproteins or fragments thereof. As is well known to those of skill in theart, the proteins according to the invention can comprise conservativeor semi-conservative substitutions, deletions or insertions wherein theprotein comprises different amino acids than those disclosed in FIG. 1and FIG. 2.

A protein of the invention can be in a substantially purified form, inwhich case it will generally comprise the polypeptide in a preparationin which more than 90%, e.g. 95%, 98%, or 99% of the polypeptide in thepreparation is a polypeptide of the invention. Proteins of the inventioncan be modified, for example by the addition of histidine residues toassist their purification or by the addition of a signal sequence topromote their secretion from a cell. Proteins having at least 90%sequence identity, for example at least 95%, 98% or 99% sequenceidentity to the polypeptide protein depicted in SEQ ID NO: 2, 4, 8 or 10may be proteins which are amino acid sequence variants, alleles,derivatives, or mutants of the protein depicted in SEQ ID NO: 2, 4, 810, and are also provided by the present invention. Similarly, proteinshaving at least 90% sequence identity, for example at least 95%, 98% or99% sequence identity to the polypeptide protein depicted in SEQ ID NO:12, 14, 16 or 18 can be proteins which are amino acid sequence variants,alleles, derivatives, or mutants of the protein depicted in SEQ ID NO:12, 14, 16 or 18, and are also provided by the present invention.

The percentage identity of protein amino acid residue sequences can becalculated by using commercially available algorithms which compare areference sequence (i.e. SEQ ID NO: 2, 4, 8, 10, 12, 14, 16, 18) with aquery sequence. The following programs (provided by the National Centerfor Biotechnology Information, NCBI) may be used to determinehomologies: BLAST, gapped BLAST, BLASTN and psi-BLAST, which may be usedwith default parameters. Use of either of the terms “homology” or“homologous” herein does not imply any necessary evolutionaryrelationship between compared sequences, in keeping with standard use ofsuch terms as “homologous recombination” which merely requires that twonucleotide sequence are sufficiently similar to recombine under theappropriate conditions.

Another method for determining the best overall match between anucleotide sequence or portion thereof, and a query sequence is the useof the FASTDB computer program based on the algorithm of Brutlag et al.,(1990, “Improved sensitivity of biological sequence database searches”Compt. Appl. Biosci., 6:237-245). The program provides a global sequencealignment. The result of such a global sequence alignment is expressedas percent identity. Suitable parameters used in a FASTDB search of anucleotide sequence to calculate the degree of identity are known.

Where a query sequence is determined to have an identity to that of SEQID NO: 2, 4, 8 or 10 of at least 90%, said sequence being that of aprotein retaining the same activity as Hmus81, such a sequence isencompassed by the present invention. Similarly, where a query sequenceis determined to have an identity to that of SEQ ID NO: 12, 14, 16 or 18of at least 90%, said sequence being that of a protein retaining thesame activity as Mmus81, such a sequence is encompassed by the presentinvention.

Preferred fragments include those comprising an epitope of the proteinsaccording to the invention. The epitopes can be determined using, forexample, peptide scanning techniques as described in the art (see e.g.Geysen et. al., 1986, “A priori determination of a peptide which mimicsa discontinuous antigenic determinant” Mol. Immunol., 23; 709-715).

The polyclonal and monoclonal antibodies according to the invention canbe produced according to techniques which are known to those skilled inthe art (e.g. Immunochemical Protocols, 2nd. edition, Pound, J. D. ed.,1998, Methods in Molecular Biology Vol. 80, Humana Press, Totowa, N.J.).For example, polyclonal antibodies can be generated by inoculating ahost animal, such as a mouse, rabbit, goat, pig, cow, horse, hamster,rat or the like, with a protein or epitope according to the inventionand recovering the immune serum. The present invention also includesfragments of whole antibodies which maintain their binding activity,such as for example, Fv, F(ab′) and F(ab′)₂ fragments as well as singlechain antibodies.

The nucleic acid and/or the proteins according to the invention can beincluded in a pharmaceutical composition together with apharmaceutically acceptable carrier, diluent or excipient therefor. Thepharmaceutical composition containing said nucleic acids according tothe invention can, for example, be used in gene therapy. Such nucleicacids, according to the invention, can be administered naked, orpackaged in protein capsules, lipid capsules, liposomes, membrane basedcapsules, virus protein, whole virus, cell vectors, bacterial cellhosts, altered mammalian cell hosts, or such suitable means foradministration.

There is further provided by the present invention a method fordetecting for the presence or absence of a nucleic acid according to theinvention, in a biological sample, which method comprises, (a) bringingsaid sample into contact with a probe comprising a nucleic acid or probeaccording to the invention under hybridizing conditions, and (b)detecting for the presence of hybridization, for example, by thepresence of any duplex or triplex formation between said probe and anynucleic acid present in said sample. Proteins according to the inventioncan also be detected by (a) contacting said sample with an antibody toan epitope of a protein according to the invention under conditionswhich allow for the formation of an antibody-antigen complex, (b)monitoring for the presence of any antigen-antibody complex.

Kits for detecting nucleic acids and proteins are also provided by thepresent invention. A kit for detecting for the presence of a nucleicacid according to the invention in a biological sample can comprise (a)means for contacting the sample with a probe comprising a nucleic acidor a probe according to the invention and means for detecting for thepresence of any duplex or triplex formation between said probe and anynucleic acid present in the sample.

Likewise, a kit for detecting for the presence of a protein according tothe invention in a biological sample can comprise (a) means forcontacting said sample with an antibody to an epitope of a proteinaccording to the invention under conditions which allow for theformation of an antibody-protein complex, and (b) means for monitoringsaid sample for the presence of any protein-antibody complex.

A further aspect of the present invention provides a method ofdetermining whether a compound is an inhibitor or an activator ofexpression or activity of the proteins of the mammalian cell cyclecheckpoint/repair pathway. The method comprises contacting a cellexpressing the proteins in said pathway with said compound and comparingthe level of expression of any of the proteins of the checkpoint/repairpathway of said cell against a cell which has not been contacted withsaid compound. Any compounds identified can then advantageously be usedas a medicament or in the preparation of a medicament for treatingcancer or proliferative disorders. Alternatively, the compounds can beincluded in a pharmaceutical composition together with apharmaceutically acceptable carrier, diluent or excipient therefor. Anycompound identified as, or any compound corresponding to a compoundidentified as an inhibitor of the cell checkpoint/repair pathway can beincluded in a pharmaceutical composition according to the inventiontogether with a cytotoxic agent, such as a DNA damaging chemotherapeuticagent, and a pharmaceutically acceptable carrier diluent or excipienttherefor. Thus, the cell cycle checkpoint/repair inhibitor can enhancethe chemotherapeutic effect of cytotoxic agents used in, for example,anti-cancer therapy.

There is also provided by the present invention a method for screeningcandidate substances for anti-cancer therapy, which method comprises (a)providing a protein according to the present invention exhibiting kinaseactivity together with a substrate for said protein under conditionssuch that the kinase will act upon the substrate, (b) bringing theprotein and substrate into contact with a candidate substance, (c)measuring the degree of any increase or decrease in the kinase activityof the protein, (d) selecting a candidate substance which provides adecrease or increase in activity. Such a candidate substance can also beused as a medicament, or in the preparation of a medicament for thetreatment of cancer or other such proliferative cell disorders.

The present invention thus provides inter alia, for therapeuticcompositions comprising (i) Hmus81 protein, fusion protein product, orbiologically active fragments thereof, (ii) nucleic acids encoding forHmus81 protein, fusion protein or fragments thereof, (iii) expressionvector constructs having an expressible nucleic acid encoding for Hmus81protein, fusion protein, or fragments thereof, (iv) anti-sense nucleicacids which correspond to the complement of nucleic acids encoding forHmus81 protein, (v) modified Hmus81 proteins, (vi) antibodies thatspecifically bind to a portion of an Hmus81 protein, (vii) transformedhost cells capable of expressing Hmus81 protein, fusion protein, orfragments thereof, and (viii) therapeutic agents identified by screeningfor the ability to bind to and/or affect the activity of Hmus81 protein.

The present invention also provides for therapeutic compositionscomprising (i) Mmus81 protein, fusion protein product, or biologicallyactive fragments thereof, (ii) nucleic acids encoding for Mmus81protein, fusion protein or fragments thereof, (iii) expression vectorconstructs having an expressible nucleic acid encoding for Mmus81protein, fusion protein, or fragments thereof, (iv) anti-sense nucleicacids which correspond to the complement of nucleic acids encoding forMmus81 protein, (v) modified Mmus81 proteins, (vi) antibodies thatspecifically bind to a portion of an Mmus81 protein, (vii) transformedhost cells capable of expressing Mmus81 protein, fusion protein, orfragments thereof, and (viii) therapeutic agents identified by screeningfor the ability to bind to and/or affect the activity of Mmus81 protein.

Therapeutic compositions of the present invention can combine mixturesof two or more species of Mus81 protein, nucleic acid encoding suchprotein, antibodies to such protein, or inhibitors of the nucleic acidtranscripts of such proteins.

A therapeutic composition of the present invention can be utilized tomake a pharmaceutical preparation for the treatment of an individual inneed of modulation of the DNA checkpoint/repair mediated by the activityof Hmus81. Another aspect of the present invention is the use of atherapeutic composition of the present invention in the formulation of apharmaceutical preparation for the treatment of an individual in need ofanti-neoplastic treatment. It is further envisioned that a therapeuticcomposition of the present invention is useful in the formulation of apharmaceutical preparation in combination with at least one otheranti-neoplastic agent for the treatment of an individual in need ofanti-neoplastic treatment.

Therapeutic compositions, or pharmaceutical formulations containing suchtherapeutic compositions, can be used to treat an individual in need ofa treatment which involves the Hmus81 mediated activity of targetedcells. Illustrative are treatment for neoplastic conditions, comprisingcontacting a cell of the individual in need of such treatment with atleast one therapeutic composition of the invention. Such therapeuticmethods can include the administration of one or more therapeuticcomposition sequentially, simultaneously, or in combination with othertherapeutics for treating a neoplastic condition.

As would be understood by one of skill in the art, many variations andequivalents to the compositions of the present invention are easilyobtained and generated through the application of routine methods knownin the art using the teaching of the present invention.

Many of the methods and materials for carrying out the basic molecularbiology manipulations as described in the examples below are known inthe art, and can be found in such references as Sambrook et al.,Molecular Cloning, 2nd edition, Cold Spring Harbor Laboratory Press(1989); Berger et al., Guide to Molecular Cloning Techniques, Methods inEnzymology, Vol. 152, Academic Press, Inc., (1987); Davis et al., BasicMethods in Molecular Biology, Elsevier Science Publishing Co., Inc.(1986); Ausubel et al., Short Protocols in Molecular Biology, 2nd ed.,John Wiley & Sons, (1992); Goeddel Gene Expression Technology, Methodsin Enzymology, Vol. 185, Academic Press, Inc., (1991); Guthrie et al.,Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology,Vol. 194, Academic Press, Inc., (1991); McPherson et al., PCR Volume 1,Oxford University Press, (1991); McPherson et al., PCR Volume 2, OxfordUniversity Press, (1995); Richardson, C. D. ed., Baculovirus ExpressionProtocols, Methods in Molecular Biology, Vol. 39, Humana Press, Inc.(1995).

The invention in its several aspects can be more readily understood withreference to the following examples.

EXAMPLE 1 HUMAN Mus81 (Hmus81) CLONING

Oligonucleotide primers Hmus81FW (GACATGGCGGCCCCGGTCCG) (SEQ ID NO: 21)and Hmus81REV (GACTCAGGTCAAGGGGCCGTAG) (SEQ ID NO: 22) corresponding tothe 5′ (ATGGCGGCCCCGGTCCG) (SEQ ID NO: 19) and 3′ (CTACGGCCCCTTGACCTGA)(SEQ ID NO: 20) ends of the putative human Mus81 ORF were used toamplify DNA products from a Marathon-Ready human cerebellum cDNA library(Clontech, Palo Alto Calif.) by polymerase chain reaction (PCR). PCR wasdone with Pfu polymerase and the following reaction conditions: 95° C.for 30″, 68° C. for 30″, 72° C. for 1-30″ (35×). The resulting DNAproducts were cloned into the pCR2.1-TOPO plasmid as recommended by themanufacturer (Invitrogen, Carlsbad Calif.) and the DNA sequenced.

Oligonucleotide primers corresponding to the 5′ and 3′ ends of Hmus81,from a putative ORF constructed using the identified yeast sequenceswere used to amplify sequences from a human cerebellum cDNA library. A1653 nucleotide sequence was obtained which encodes a 551 amino acidprotein (SEQ ID NO: 2) with significant similarity to the yeast Mus81sequences (SEQ ID NO: 5). A longer 1857 nucleotide sequence encodes fora shorter variant of Hmus81 that is a 455 amino acid protein (SEQ ID NO:4). This results from the presence of a stop codon within a DNA insertfrom position 1274 to 1474 of the nucleotide sequence (SEQ ID NO: 25).

EXAMPLE 2 MOUSE Mus81 CLONING

Oligonucleotide primers RJH030 (GAGACTCTGAAGGAGCCAG) (SEQ ID NO: 23) andRJH031 (GCTAAAAGGCTAGCCAGCC) (SEQ ID NO: 24) corresponding to sequencesflanking the 5′ and 3′ ends of the putative mouse Mus81 ORF were used toamplify DNA products from a Marathon-Ready mouse brain cDNA library(Clontech) by PCR. The following conditions were used: 95° C. for 60″,60° C. for 60″, 72° C. for 2′30″ (35×). The resulting PCR products werecloned into the pCR2.1-TOPO plasmid (Invitrogen) and the DNA sequenced.

The human cDNA sequences were used to search for homologous mousesequences in the public databases. Several ESTs with significanthomology to the 5′ and 3′ ends of the human sequence were identified.This resulted in the amplification of several sequences (probablyrepresenting splicing variants) encoding proteins from 424 to 551 aminoacids (FIG. 2).

The translation products of the human and mouse cDNAs have significantsimilarity to the yeast Mus81 amino acid sequences. The longest human(Hmus81₄) and mouse (Mmus81) translation products are 17-20% identicaland 30-40% similar to the yeast proteins. No other mammalian proteinshad high similarity with the yeast proteins indicating that this hadidentified the closest homologues. The mouse sequence is 81% identicaland 87% similar to the human protein. Alignment of the mammalian andyeast proteins demonstrates that there is similarity throughout, withmore highly conserved regions in the central and C-terminal regions ofthe proteins (FIG. 3). The conserved central region is found in the XPFfamily of endonucleases and corresponds to the catalytic site (Aravindet al., “Conserved domains in DNA repair protein and evolution of repairsystems” Nucleic Acids Res 27(5): 1223-1242, 1999).

EXAMPLE 3 NORTHERN BLOT HYBRIDISATION

Human multiple tissue and cancer cell line blots (Clontech) werehybridized with a 1.7 kb probe corresponding to human Mus81 cDNA usingthe QuickHyb method as described by the manufacturer (Clontech). Theblots were washed at high stringency (0.1×SSC, 0.1% SDS, 50° C., 2×20min) and signals were detected by autoradiography.

Northern blot analysis using the Hmus81 cDNA as probe demonstrated thatspecific transcripts of approximately 2.5-3.0 kb were present in mosthuman tissues with lower levels in lung, liver and kidney (FIG. 6).

EXAMPLE 4 IDENTIFICATION OF A Cds1 FHA DOMAIN-BINDING PROTEIN

A yeast two-hybrid screen was employed using the S. pombe Cds1 FHAdomain as bait and a S. pombe cDNA library as prey. Transformants thatgrew in the selection conditions for interaction between the bait andprey proteins were isolated and tested in secondary screens forspecificity of interaction. One of the transformants that was isolatedfrom this screen contained a cDNA sequence that encoded a 572 amino acidhypothetical protein (PID g2213548). The amino acid sequences encoded bythe S. pombe ORF SPCC4G3.05c (Spmus81) (SEQ ID NO:6) and S. cerevisiaeORF YDR386W (ScMus81) (SEQ ID NO:5) were compared and alignment of thetranslation products of the yeast and human sequences for amino acidsequence comparison was performed with the program CLUSTALW.

The translation product of this S. pombe ORF (mus81⁺) was found to havesignificant homology to the S. cerevisiae hypothetical protein encodedby ORF YDR386w (25% identity, 42% similarity). This protein has beenannotated as Mus81 in the Saccharomyces Genome Database and is reportedto be in a complex with the DNA repair protein Rad54. A null mutant isreported to be viable, but defective in meiosis and sensitive to the DNAdamaging agents MMS and UV light. The genomic copy of S. pombe mus81⁺was tagged at the 3′ end with three tandem copies of the haemoinfluenzaHA epitope through site-directed recombination.

Antibodies directed against the HA epitope detected polypeptides from anasynchronous culture which migrated through SDS-PAGE with a mobility ofapproximately 65-70 kDa. The calculated predicted molecular weight beingabout 65 kDa.

The presence of multiple polypeptides demonstrates that the protein canbe post-translationally modified, possibly by phosphorylation. Theproportion of slower migrating polypeptides in asynchronous cultures wasincreased by treatment of the cells with hydroxyurea, a ribonucleotidereductase inhibitor that causes a cell cycle arrest in S-phase. Thisshows that the post-translational modification is cell cycle regulated,and may be checkpoint dependent. The increased modification of Mus81 wasnot observed in Cds1 and Rad3 checkpoint mutant strains, but did occurin a rad54 mutant strain. The physical interaction between Cds1 andMus81 was confirmed in vivo by co-immunoprecipitation of the twoproteins.

Inactivation of Mus81 makes fission yeast more sensitive to UVirradiation. This is also observed in yeast strains that are defectivefor the two repair pathways that account for all detectable repair of UVinduced damage (nucleotide excision repair and UV excision repair). Thissuggests that Mus81 is required for tolerating UV damage.

In order to determine whether the product encoded by this gene isinvolved in checkpoint/repair responses, a S. pombe strain was generatedin which the entire ORF for mus81 was deleted by site-directedrecombination. This mutant strain had increased sensitivity to UVirradiation, but appeared to have an intact checkpoint/repair responsein the presence of DNA damage.

EXAMPLE 5 INTERACTION BETWEEN HUMAN Mus81 and Cds1

The Hmus81, ORF was cloned into the mammalian transient expressionvector pYC1HA (Fu et al., “TNIK, a novel member of the germinal centerkinase family that activates the c-Jun N-terminal kinase pathway andregulates the cytoskeleton” J. Biol. Chem. 274(43):30729-30737, 1999)immediately downstream of and in frame with the HA epitope tag.Similarly, the human Cds1 ORF was cloned into the pYC1FLAG (Fu et al.,supra 1999) expression vector downstream of and in frame with the FLAGepitope. Plasmid DNA was used to transfect HEK293 cells using Superfectreagent as described by the manufacturer (Qiagen). After 24 hours, thecells were collected in lysis buffer (1% NP40, 50 mM Tris HCl pH 7.5,150 mM NaCl, 1 mM DTT) supplemented with Pefabloc®SC and Complete™protease inhibitors as recommended by the manufacturer (BoehringerMannheim). The lysates were cleared of debris by centrifugation at 10000g for 15 min. (4° C.).

Cleared supernatants from cells transiently expressing epitope-taggedproteins were incubated several hours at 4° C. with agarose bead-linkedantibodies directed against the HA (Santa Cruz Biotechnologies) or FLAG(OctA-probe™, Santa Cruz Biotechnologies) epitope. The agarose beadswere then washed 3 times with lysis buffer, resuspended in SDSdenaturing buffer and incubated at 95° C. for 5 min. Supernatants andimmunoprecipitates were resolved by SDS-PAGE and transferred to PVDFmembranes. The membranes were blocked with TTBS (150 mM NaCl, 100 mMTris-HCl pH7.5, 0.1% Tween 20) containing 5% skimmed milk. For detectionof HA-tagged Mus81 protein, the blots were incubated for two hours atroom temperature with horseradish peroxidase conjugated anti-HAantibodies (Santa Cruz Biotechnologies) diluted to 1:1000 in TTBScontaining 0.1% milk. For detection of FLAG-tagged Cds1, blots wereincubated with anti-FLAG® M2 antibody (Sigma) diluted to 1:3000 in TTBS.The blot washed with TTBS and then incubated for 1 hour at roomtemperature with horseradish peroxidase conjugated anti-mouse Igantibody diluted to 1:3000 in TTBS. Finally, the blots were washed withTTBS and signals detected using the ECL-Plus chemoluminescence detectionsystem as described by the manufacturer (Amersham Pharmacia Biotech).

In order to determine whether human Mus81 and Cds1 are capable ofinteracting, the proteins were tagged with the HA and FLAG epitopes,respectively, and expressed transiently in mammalian cells alone or incombination. Cell lysates were prepared from the transfected cells andimmunoprecipitations were carried out with antibodies against theepitope tags. The resulting immunoprecipitates were subjected to westernblot analysis with the reciprocal antibody. The HA antibodies recognizeda 65 kDa protein, the expected size for the tagged version of humanMus81, only in lysates from cells transfected with the Mus81 construct.Similarly, the FLAG antibodies recognized a 65 kDa protein correspondingto tagged Cds1 only in lysates from cells expressing Cds1-FLAG (FIG. 8).Mus81 was also detected in precipitates obtained with the FLAG antibodyfrom lysates of cells transfected with both Cds1 and Mus81. However,Mus81 was not present in precipitates from cells expressing only Mus81or Cds1. Conversely, Cds1 was only detected in precipitates obtainedwith the HA antibody from cells expressing both tagged proteins. Theseresults indicate that the human Mus81 and Cds1 proteins are capable ofinteracting in mammalian cells. This suggests that the Hmus81 protein isinvolved in UV DNA damage repair.

The present invention identifies the human and mouse homologues of theyeast Mus81 protein, which are involved in UV damage tolerance andinteracts with the FHA domain of fission yeast Cds1. Human Mus81 ispresent as various splicing isoforms and is expressed in most humantissues and cancer cell lines. Analysis of a Mus81-GFP fusion proteinsuggests that it is predominantly nuclear while co-immunoprecipitationof tagged forms of human Mus81 and Cds1 indicate that they form acomplex in mammalian cells.

EXAMPLE 6 GENOMIC STRUCTURE AND CHROMOSOMAL LOCALIZATION OF HUMAN Mus81

The human cDNAs were used to identify contiguous genomic sequencescontaining Mus81 in the public databases. Comparison of the genomicsequence confirmed that the various cDNA forms corresponded to differentsplice variants. Examination of the results identified 18 exons encodingMus81 sequences within a 5.8 kb genomic region (FIG. 4). The splicingdifferences in the cDNAs identified occurred in the region encompassingexons 13 and 14. The nucleic acid encoding for human Mus81₂ (SEQ IDNO:3) was composed of all of the exons identified. The nucleic acidencoding for human Mus81₁ (SEQ ID NO: 1) did not contain exon 13 and thenucleic acid encoding for human Mus81₃ (SEQ ID NO: 7) was lacking exons13 and 14. Splicing of the nucleic acid encoding for human Mus81₄ (SEQID NO: 9) was identical to that found in the nucleic acid encoding forhuman Mus81₁, (SEQ ID NO: 1) except that it contained three additionalnucleotides (CAG) at the 5′ end of exon 14 due to utilization of analternative splice acceptor site. Splicing of all introns utilized theconsensus donor and acceptor sites.

Fluorescence in situ Hybridisation (FISH) analysis was carried out usingstandard procedures. Briefly, human lymphocytes isolated from blood weresynchronized by culturing in the presence of 0.18 mg/ml BrdU. The BrdUwashed off to release the block and the cells were cultured for 6 hoursprior to harvesting and fixation. FISH detection was carried out with aMus81 cDNA probe labelled with biotinylated dATP. Chromosomallocalization was determined by comparison of FISH signals to DAPIbanding pattern.

FISH analysis using human Mus81 cDNA as a probe resulted in staining ofa single pair of chromosomes at 11q13 in 70 out of 100 mitotic spreads(FIG. 5). This localization was confirmed by the previous assignment ofa public EST (WI-18484), which is identical to part of the Mus81sequence, to chromosome 11 on the WICGR radiation hybrid map.

EXAMPLE 7 EXPRESSION AND INTRACELLULAR LOCALIZATION OF HUMAN Mus81

The human Mus81₄ cDNA was cloned downstream and in frame with the greenfluorescent protein (GFP) encoding open reading frame gene (ORF) in aretrovirus expression vector. The retrovirus expression vector is chosento allow for the regulated expression of proteins of interest, and in apreferred embodiment allows fusion of the protein of interest to the GFPor modified GFP for visualization of expression. It is also possible toexpress both the Mus81 protein and GFP protein as separate proteins fromthe same expression vector.

Commercially available vectors suitable for expression of Mus81 proteininclude and are not limited to, for example, pRevTRE (Clontech) whichare derived from the pLNCX (Clontech) retroviral expression vector(Gossen, M. & Bujard, H., 1992, “Tight control of gene expression inmammalian cells by tetracycline-responsive promoters” PNAS(USA)89:5547-5551), or GFP fusion protein expressing retroviral expressionvectors pLEGFP-N1 and pLEGFP-C1 (Clontech).

The Human Mus81-GFP expressing retrovirus vector was used to infect A549lung carcinoma cells containing an integrated copy of the tTAtransactivator for regulated expression of the fusion protein. The cellswere grown to allow expression of the fusion protein, and visualized byfluorescence microscopy three days after infection.

Human Mus81 was expressed as a fusion with the GFP protein in A549cells. Fluorescence was detected primarily in the nuclei of these cells(FIG. 7). The nuclear localization of Hmus81 is in agreement with itsrole in DNA repair associated functions.

The invention, having been fully described in many of its aspects andclaimed herein can be made and executed without undue experimentation byone of skill in the art according to the teaching herein. While thecompositions and methods of this invention have been described by way ofexample above, it will be apparent to those of skill in the art thatmany variations and modifications can be applied to the compositions andmethods described herein without departing from the concept, spirit andscope of the invention.

1. An isolated mammalian Mus81 protein.
 2. The isolated protein of claim1 which is either a human Mus81 protein or murine Mus81 protein.
 3. Theisolated Mus81 protein of claim 1, comprising a human protein selectedfrom the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 8,and SEQ ID NO:10.
 4. A fusion protein comprising a protein of claim 1 orbiologically active portion thereof.
 5. The fusion protein of claim 4identifiable as Hmus81-GFP.
 6. An antibody which specifically binds to aportion of a protein of claim
 3. 7. The isolated Mus81 protein of claim1, comprising a murine protein selected from the group consisting of SEQID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, and SEQ ID NO:18.
 8. Anantibody which specifically binds to a portion of a protein of claim 7.9. A method for identifying a compound as an inhibitor or activator ofexpression of the mammalian Mus81 cell cycle checkpoint/repair pathwayprotein of claim 1, which method comprises contacting a candidatecompound with a cell expressing the protein in said pathway andcomparing the level of expression of the human Mus81 human cell cyclecheckpoint/repair pathway protein in said cell with that of a cell whichhas not been contacted with said candidate compound.
 10. A method foridentifying a compound as an inhibitor or activator of expression of themammalian Mus81 cell cycle checkpoint/repair pathway protein of claim 3,which method comprises contacting a candidate compound with a cellexpressing the protein in said pathway and comparing the level ofexpression of the human Mus81 human cell cycle checkpoint/repair pathwayprotein in said cell with that of a cell which has not been contactedwith said candidate compound.
 11. A method of increasing susceptibilityof cancer cells to chemotherapy or radiotherapy, which method comprisesadministering to a patient a therapeutically effective amount of anantisense nucleic acid comprising a nucleic acid sequence that is thecomplement of at least a portion of a nucleic acid encoding the Mus81protein of claim
 1. 12. A method of increasing susceptibility of cancercells to chemotherapy or radiotherapy, which method comprisesadministering to a patient a therapeutically effective amount of anantisense nucleic acid comprising a nucleic acid sequence that is thecomplement of at least a portion of a nucleic acid encoding the Mus81protein of claim
 3. 13. A method of increasing susceptibility of cancercells to chemotherapy or radiotherapy, which method comprisesadministering to a patient a therapeutically effective amount of aninhibitor compound that inhibits the activity of the Mus81 protein ofclaim
 1. 14. A method of increasing susceptibility of cancer cells tochemotherapy or radiotherapy, which method comprises administering to apatient a therapeutically effective amount of an inhibitor compound thatinhibits the activity of the Mus81 protein of claim
 3. 15. A method ofidentifying a chemical compound that modulates Mus81 dependent cellcycle pathway, which method comprises administering a chemical compoundto be tested to a host cell, detecting the level of the Mus81 protein ofclaim 1, or a subsequent cell cycle protein in said cell, and comparingthe detected level to that of a normal untreated cell.
 16. A method ofidentifying a chemical compound that modulates Mus81 dependent cellcycle pathway, which method comprises administering a chemical compoundto be tested to a host cell, detecting the level of the Mus81 protein ofclaim 3, or a subsequent cell cycle protein in said cell, and comparingthe detected level to that of a normal untreated cell.
 17. A method ofidentifying a chemical compound that modulates Mus81 dependent cellcycle pathway, which method comprises administering a chemical compoundto be tested to a host cell, detecting the level of a nucleic acidencoding the Mus81 protein of claim 1, or encoding a subsequent cellcycle protein in said cell, and comparing the detected level to that ofa normal untreated cell.
 18. A method of identifying a chemical compoundthat modulates Mus81 dependent cell cycle pathway, which methodcomprises administering a chemical compound to be tested to a host cell,detecting the level of a nucleic acid encoding the Mus81 protein ofclaim 3, or encoding a subsequent cell cycle protein in said cell, andcomparing the detected level to that of a normal untreated cell.
 19. Anisolated nucleic acid encoding for a murine Mus81 protein or abiologically active fragment thereof.
 20. The nucleic acid of claim 19comprising a nucleotide sequence selected from the group consisting of(a) residues 42-1694 of SEQ ID NO: 11, (b) residues 15-1323 of SEQ IDNO: 13, (c) residues 52-1644 of SEQ ID NO: 15, and (d) residues 52-1614of SEQ ID NO:
 17. 21. The nucleic acid of claim 19, wherein said nucleicacid encodes for a murine Mus81 protein comprising the amino acidsequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO:14, SEQ ID NO: 16, and SEQ ID NO:
 18. 22. An expression vectorcomprising a nucleic acid of claim
 20. 23. A host cell transformed witha vector of claim
 22. 24. An expression vector comprising a nucleic acidof claim
 21. 25. A host cell transformed with a vector of claim 24.